In eukaryotes, sugar residues are commonly linked to four different amino acid residues. These amino acid residues are classified as O-linked (serine, threonine, and hydroxylysine) and N-linked (asparagine). The O-linked sugars are synthesized in the Golgi or rough Endoplasmic Reticulum (ER) from nucleotide sugars. The N-linked sugars are synthesized from a common precursor, and subsequently processed. It is known that addition of N-linked carbohydrate chains is important for stabilization of folding, prevention of degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins. The addition of N-linked oligosaccharides to specific Asn residues plays an important role in regulating the activity, stability or antigenicity of mature proteins of viruses (Opdenakker G. et al FASEB Journal 7, 1330-1337 1993). It has also been suggested that N-linked glycosylation is required for folding, transport, cell surface expression, secretion of glycoproteins (Helenius, A., Molecular Biology of the Cell 5, 253-265 1994), protection from proteolytic degradation and enhancement of glycoprotein solubility (Doms et al., Virology 193, 545-562 1993). Viral surface glycoproteins are not only required for correct protein folding, but also provide protection against neutralizing antibodies as a “glycan shield.” As a result, strong host-specific selection is frequently associated with codon positions of potential N-linked glycosylation. Consequently N-linked glycosylation sites tend to be conserved across strains and clades.
There are three main types of influenza virus: A, B and C. Type A strains of influenza virus can cause severe illness and are the only type to have caused human pandemics. The H5N1 strain is a type A influenza virus. Type B strains cause sporadic human cases and small-scale outbreaks. Type C strains only rarely cause human infection and have not caused large outbreaks. Of the influenza A viruses, only subtypes H1, H2 and H3 have been transmitted easily between humans.
Outbreaks of influenza A virus continue to cause widespread morbidity and mortality worldwide. In the United States alone, an estimated 5 to 20% of the population is infected by influenza A virus annually, causing approximately 200,000 hospitalizations and 36,000 deaths. The establishment of comprehensive vaccination policies has been an effective measure to limit influenza morbidity. However, the frequent genetic drifting of the virus requires yearly reformulation of the vaccine, potentially leading to a mismatch between the viral strain present in the vaccine and that circulating. Thus, antiviral therapies against influenza virus are important tools to limit both disease severity as well as transmission.
The highly pathogenic H5N1 influenza viruses have caused outbreaks in poultry and wild birds since 2003 (Li K S et al. (2004) Nature 430:209-213). As of February 2010, these viruses have infected not only avian species but also over 478 humans, of which 286 cases proved to be fatal (www.who.int/esr/disease/avian_influenza/country/cases_table—2010—02—17/en/index.html). The highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (Garten R J, et al. (2009) Science 325:197-201, Neumann G, et al. (2009) Nature 459:931-939). There is great concern that an influenza virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan.
Influenza viruses are segmented negative-strand RNA viruses and belong to the Orthomyxoviridae family. Influenza A virus consists of 9 structural proteins and codes additionally for one nonstructural NS1 protein with regulatory functions. The non-structural NS1 protein is synthesized in large quantities during the reproduction cycle and is localized in the cytosol and nucleus of the infected cells. The segmented nature of the viral genome allows the mechanism of genetic reassortment (exchange of genome segments) to take place during mixed infection of a cell with different viral strains. The influenza A virus may be further classified into various subtypes depending on the different hemagglutinin (HA) and neuraminidase (NA) viral proteins displayed on their surface. Influenza A virus subtypes are identified by two viral surface glycoproteins, hemagglutinin (HA or H) and neuraminidase (NA or N). Each influenza virus subtype is identified by its combination of H and N proteins. There are 16 known HA subtypes and 9 known NA subtypes. Influenza type A viruses can infect people, birds, pigs, horses, and other animals, but wild birds are the natural hosts for these viruses. Only some influenza A subtypes (i.e., H1N1, H1N2, and H3N2) are currently in circulation among people, but all combinations of the 16H and 9 NA subtypes have been identified in avian species, especially in wild waterfowl and shorebirds. In addition, there is increasing evidence that H5 and H7 influenza viruses can also cause human illness.
The HA of influenza A virus comprises two structurally distinct regions, namely, a globular head region and a stem region. The globular head region contains a receptor binding site which is responsible for virus attachment to a target cell and participates in the hemagglutination activity of HA. The stem region contains a fusion peptide which is necessary for membrane fusion between the viral envelope and an endosomal membrane of the cell and thus relates to fusion activity (Wiley et al., Ann Rev. Biochem., 56:365-394 (1987)).
Important contributions to the understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (Kuiken T, et al. (2006) Science 312:394-397; Maines T R, et al. (2009) Science 325:484-487; Skehel J J, Wiley D C (2000) Ann Rev Biochem 69:531-569; van Riel D, et al. (2006) Science 312:399-399). To cross the species barrier and infect the human population, avian HA must change its receptor-binding preference from a terminally sialylated glycan that contains α2,3 (avian)-linked to α2,6 (human)-linked sialic acid motifs (Connor R J, et al. (1994) Virology 205:17-23), and this switch could occur through only two mutations, as in the 1918 pandemic (Tumpey T M, et al. (2007) Science 315:655-659). Therefore, understanding the factors that affect influenza binding to glycan receptors is critical for developing methods to control any future crossover influenza strains that have pandemic potential.
To address the need for making a candidate influenza vaccine that could induce potent neutralizing antibodies against divergent strains of H5N1 influenza viruses a consensus H5N1 hemagglutinin (HA) sequence based vaccine elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 clades. (Chen M W, et al. (2008) Proc Natl Acad Sci USA 105:13538-13543).
HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (Kuiken T, et al. (2006) Science 312:394-397). Both regions carry N-linked oligosaccharides (Keil W, et al. (1985) EMBO J 4:2711-2720), which affect the functional properties of HA (Chen Z Y, et al. (2008) Vaccine 26:361-371; Ohuchi R, et al. (1997) J Virol 71:3719-3725). Among different subtypes of influenza A viruses, there is extensive variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (Ohuchi R, et al. (1997) J Virol 71:3719-3725). Glycans near antigenic peptide epitopes interfere with antibody recognition (Skehel J J, et al. (1984) Proc Natl Acad Sci USA 81:1779-1783), and glycans near the proteolytic activation site of HA modulate cleavage and influence the infectivity of influenza virus (Deshpande K L, et al. (1987) Proc Natl Acad Sci USA 84:36-40). Mutational deletion of HA glycosylation sites can affect viral receptor binding (Gunther I, et al. (1993) Virus Res 27:147-160).
Changes in the peptide sequence at or near glycosylation sites may alter HA's 3D structure, and thus receptor-binding specificity and affinity. Indeed, HAs from different H5N1 subtypes have different glycan-binding patterns (Stevens J, et al. (2008) J Mol Biol 381:1382-1394). Mutagenesis of glycosylation sites on H1 and H3 has been studied in the whole-viral system (Chandrasekaran A, et al. (2008) Nat Biotechnol 26:107-113; Deom C M, et al. (1986) Proc Natl Acad Sci USA 83:3771-3775). However, it is not known how changes in glycosylation affect receptor-binding specificity and affinity, especially with regard to the most pathogenic H5N1 HA.
Flu vaccines, when made, have to be changed every year as the less highly glycosylated or non-glycosylated regions of hemagglutinin continue to mutate to escape from the host immune system.
The goal of vaccine design against heterogeneous pathogens is to identify and design effective and broadly protective antigens. In the case of influenza, considerable historical efforts have gone into the empirical testing of conserved linear sequences and regions with little success. A plausible reason for these failures is a lack of knowledge that focused responses against antigenic test articles are actual bona fide productive sites for neutralization of an antigen on the pathogen in the setting of an actual infection.